The 24K estrogen-regulated protein was discovered by our laboratory in MCF-7 human breast cancer cells, and a monoclonal antibody was prepared. We have also prepared and partially sequenced a 24K cDNA, and found a tentative homology with the low molecular weight heat shock proteins. The 24K protein is found to be concentrated in cytoplasmic vacuoles in the cells of a number of normal female reproductive tissues and in some endometrial tumors, and also in many but not all breast tumors. In breast tumors, its presence is correlated with that of estrogen and progesterone receptors, suggesting its possible utility as a marker of differentiation and hormone responsiveness. We now propose three studies to examine the biological and clinical significance of 24K in breast tumor cells. (1) We will determine the complete 24K cDNA nucleotide sequence, search for confirmation of homology with known proteins including the heat shock proteins, and examine its possible regulation by stress factors as well as by steroid hormones. (2) We will use 24K, as an estrogen-regulated protein, to study tumors with discordant ER and PgR status. Tumors in which 24K is discordant with receptor status will also be examined. Specific molecular alteration in ER, PgR, or 24K itself which might be predicted by each of these discordances will be tested at several levels, and the relationship of each type of discordance with clinical outcome will be defined. (3) We will determine whether 24K has useful independent prognostic value, by assaying 24K in breast tumor specimens from our Tumor Bank for which clinical outcome and many known prognostic factors (ER and PgR, nodal status, DNA S-phase and ploidy, HER-2/neu oncogene amplification) are available for correlation. We will also assay 24K in parallel with the other factors in new breast tumor specimens, for multivariate analysis of their association with recurrence and survival as follow-up clinical becomes available. These studies will determine the functional identity of 24K, its ability to discriminate good and bad prognosis subgroups in breast tumors with discordant ER and PgR, and its role as an independent prognostic factor for selecting optimum treatment strategies for individual breast cancer patients.